O.I. Panasenko, V.V. Parchenko, A.S. Gotsulya, I.V. Melnik, A.A. Safonov, V.A. Salionov, R.A. Scherbyna, V.P. Buryak, N.A. Postol, T.O. Samura, I.M. Keithyn, S.N. Kulish, Jul. Timoshyk, Panasenko T.V.

Zaporozhye State Medical University

APPLICATION OF THE CHROMATOGRAPHY IN FORENSIC TOXICOLOGY

The father of chromatography is recognized as the Russian botanist M.S.Tswett. He stated the "chromatography" is a method in which the components of a mixture are separated on an adsorbent column in a flowing system. In his original experiments (1903), Tswett packed a fine powder such as sucrose into a glass tule to produce a column of the desired height. After extracting green and yellow chloroplast pigments from leaves and transfecting them to petroleum ether, he poured a small volume of the extract onto the column. When the pigments had formed a narron initial band at the top of the adsorbent, fresh solvent (petroleum ether ) was added and pressure applied to the top of the column. As the solvent flowed through the, column the individual pigments move at different rates and were eventually separated from each other. The key features of Tswett's technique where the application of the mixture as a narrow initial zone and the development of the chromatogram by application of fresh solvent. Others had emploed procedures based on the phenomena of adsorption or partition, but these lacked Tswett's critical development step and therefore did not yield effective resolution of mixtures.

Amongst many subsequent developments. those A.I.P. Martin (1910-2002) and R.L.M. Synge (1914-1994), who were awarded the 1952 Nobel Prize in Chemistry for the discovery of partition chromatography [9] and of A.T.James and A.I.P. Martin who developed gas-liquid partition chromatography [6], stand out [1]. Further important work lay in the development of sensitive detector for GC.The flame-ionization detector (FID) responds to most organic compounds,whilst the electron-capture detector (ECD) shows an enhanced and selective response to compounds containing strong electronegative moieties such as halogen atoms or nitro groups. The nitrogen-phosphorus detector (N>P>D) also known as the alkali flame-ionization detector (AFID), shows an enhanced and selective response to compounds containing C-N bonds of phosphorus . In 1948 the Swedish analyst A.W.K. Tiselius was awarded the Nobel Prize in Chemistry for his pionering work in developing electrophoresis. Capillary electrophoresis (CE) was pioneered by Hjerten, who had built tube electrophoresis units by 1959 and went on to demonstrate that free-flowing electrophoresis in capillary tubes with UV detection was feasible.

Forgeston and Lukacs [7] developed the first truly useful CE instrument and showed that it had exceptional resolution. More resently, capillary electrophoresis and related techiques have been used in the analysis of drugs and other poisons.

Chromatography and electrophoresis have developed into a range of modes including paper chromatography (PC), thinlayer chromatography (TLC), ion-exchange chromatography, gel-permeation chromatography (GPC, also known as size-exclusion chromatography, SEC), affinity chromatography, gas-chromatography (GC), supercritical-fluid chromatography (SFC), high-performance chromatography (HPLC), capillary electrochromatography (CEC). CE and CEC are hydrin techniques whereas eluent flow is produced by electro-osmosis.

In the 1950s there was great emphasis on the development of TLC methods. At the same time, research in liquid chromatography led to the development of various amino-acid analyzers. These analyzers used columns made of styrene/ divinylbenzene polymers that were derivatized to make strong cation-exchange (SCX) materials.After gradient elution (changing the eluent composition with time in a defined way) , the column eluate was mixed with ninhydrin, heated and passed through a spectrophotometric cell and the absorbance monitored (570 nm). The analysis of one sample could take 2 days. In PC and TLC, techniques that are sometimes referred to as planar chromatography or development chromatography, solutions of the analytes are usually applied as small discrete spots or bands. The application solvent is evaporated before the edge of the paper sheet or strip, or thin-layer plate, is placed in the liquid mobile phase, which is drawn along the sheet or plate ( the stationary phase ) by capillary y action. Several analyses may be performd in parallel. All the analytes are detected ( narmally visualized ) at the end of the development process.

In most other form of chromatography, a sample is either added to the eluent, which may be a gas, a liquid or a supercritical fluid, such as carbon dioxide, or placed on a support material or otherwise concentrated before the eluent is introduced. The eluent containing the analyte and other components of the saample/ sample extract is then allowed or made to flow through or past a stationary phase supported within a column. The mobile and stationary phases are chosen such that different components of the sample different affinities for each phase. A component that has poor affinity for the stationary phase will pass through the column quite quickly, and vice versa. As a result f these differences in mobility, sample components became separated as they travel through the column. This process is called elution chromatography and analytes are detected sequentially as they elute from the column. In both development and elution chromatography sample transport is by continuous addition of mobile phase. Various modifications of these techniques are possible, for example development of a TLC plate in a second dimension using a different mobile phase.

There has been continuous development in many branches chromatography and in CE since 1960s, particularly in materials and in the refinement of instrumentation that has resulted in the efficient, reliable and sensitive analytical methods that form the backbone of modern routine laboratory analysis [2,4,5]. Manufacturers catalogues and wed, sites often contain up-to-date information on newer products, although important experimental details may be lacking. Chromatograms, for example, may have been injected; in some cases the the actual amount injected and the detector sensitivity may not be stated. As well as the formal scientific literature, the trend to publications sponsored by manufacturers or funded from advertising has produced same useful free magazines, LC GC is probably the best of these.

Chromatographic theory was studied by wilson [11], who discussed the quantitative aspects of chromatography in terms of diffusion, rate of adsorphion, and isotherm nonlinearity.The first comprehensive mathematical treatment describing column performance in terms of stationary phase particle size and diffusion , was presented by Martin [10]. However, it was van Deemter et al [12] who developed the rate theory to describe the separation processes following on from earlier work of Lapidus and Admunson. Gidding and Eyring [3] first looked at the chromatography and from the 1960s onwards, examined many aspects of GC and general chromatography. In the simplest forms of GC and of HPLC , the stationary phase is simply a rigid material packed within a column through which the eluent flows, but more usually the stationary phase is coated or bonded directly to a column, or to particles of a rigid support material packed within the column. In the mid 1960s, discussion of the parallels between LC and GC suggested that use of smaller particles in HPLC would lead to better efficiency, hence greater speed of analysis, and this better sensitivity/selectivity. The problem with columns packed with larger particles was the slow mass transfer of the analyte molecules into and out of the pores of the stationary material packing made with smaller particles were thus investigated to improve resolution. A range of suitably small particle size packing based on 10 mm average particle size silica gels were soon introduced, and work began on how best to pack these small particles to give efficient columns.

Retention factors (k), absolute retention times (volumes) and retention times relative to the retention times relative to the retention of a given campound (internal standard) can be useful ways of recording retention data in GC. However, the Kovats retention index [8] provides a methodvof recording retention data that is independent of eluent flow rate, column length, phase leading and operating temperature. Moveorer, accurate measurement of to is not required. Straight-chain hydrocarbons are assigned an index of 100 x the number of carbon atoms in the molecule )e.g. decane =1000). The retention index of a given analyte at a given column temperature is then calculated by difference from the retention indices of the normal alkanes eluting before and after the analyte. Retention indices can also be calculated from data generated on a temperature program applying the following formula during individual ramps of the program: RI=

where: RI=retention index of x.z=n-alkane with z carbon atoms eluting before x, trx=retention time of x,trz=retention time of z and tr(z+1)= retention time of n-alkane with z+1 carbon atoms eluting after x Many attempts have been made to develop a suitable retention index system for HPLC using for example, homologous series of alcohols, ketones or nitroalkanes, but in practice such methods often no advantage over retention factor and other simpler ways of expressing retention in HPLC .

SUMMARY

Chromatography techniques, notably GC and HPLC, and to an extent TLC, are of unrivalled importance in analytical toxicology as discussed in the following chapters, but must be used with due care and attention to detail if reliable results are to be obtained. An appreciation of the theoretical aspects of chromatography as presented here is important in making best use of the resolving power or these systems. Hower, SFC, once widely advocated for a variety of applications, has been largely discarded.Capillary electrophoretic techniques too, whilst of value in pharmaceutical QC and in separating enantiomers on an analytical scale , at present have neither the sensitivity nor the mechanical strength to provide a robust system for brass analysis.

REFERENCES

1. Adlard E.R. Fifty years of gas chromatography/E.R. Adlard//Chromatographia, 2003.-val.57.-pp 13-18

2. Ettre L.S. Milestones in the Evolution of Chromatography/L.S. Ettre// ChromSource, Franklin. T.N, 2002.-624p.

3. Gidding I.C. A olecular dynamic theory of chromatography/ I.C. Giddings, H.Eyring//I.Phys.Chem., 1955.val.59.-pp. 416-421

4. Grob R.I. Modern Practice of Gas Chromatography/R.I.Grob, EF. Barry//Wiley, 4-th edn., New York, 2004.-676p.

5. Issad H.Y. A century of Separation Science/ H.I. Issad//Dekker, New York,2002.-735p.

6. Fames A.T. Gas-liquid portion chromatography: the separation and microestimation of volatile fatty acid from formic acid to dodecanoic acid./ A.T.Fames, A.I.P. Martin // Biochem. I. 1952.-val.50.-pp.675-690

7. Forgenson I.W. Free-zone electrophoresis in glass capillaries/I.W. Forgenson, K.D. Lukas//Clin.Chem., 1981.-val.27.-pp.1551-1553

8. Kovats E. Zusammenhange zwischen sructur und gaschromatographischen daten organischer verbindungen/E.Kozats//Fresenium Z. Anal. Chem., 1961 H.181.-S. 351-366

9. Martin A. I. P. A new form of chromatogram involving two liquid phases/ A.I.P. Martin, R.L.M.Synge//Biochem.I., 1941.-val.35.-pp.1358-1368

10. Some theoretical aspects of partion chromatography/ A.I.P. Martin// Biochem. Sac. Symp., 1949.-val.3.-pp.4-20

11. Van Deemterm I.I. Longitudinal diffusion and resistance to mass transfer as cause of nonideality in Chromatography/I.I.Deemter.F.I. Zuiderweg.A. Klinkenberg//Chem.Eng. Sci., 1956.val.3.-pp 271-289